Development of light response and GABAergic excitation-to-inhibition switch in zebrafish retinal ganglion cells

Abstract

The zebrafish retina has been an important model for studying morphological development of neural circuits in vivo. However, its functional development is not yet well understood. To investigate the functional development of zebrafish retina, we developed an in vivo patch-clamp whole-cell recording technique in intact zebrafish larvae. We first examined the developmental profile of light-evoked responses (LERs) in retinal ganglion cells (RGCs) from 2 to 9 days post-fertilization (dpf). Unstable LERs were first observed at 2.5 dpf. By 4 dpf, RGCs exhibited reliable light responses. As the GABAergic system is critical for retinal development, we then performed in vivo gramicidin perforated-patch whole-cell recording to characterize the developmental change of GABAergic action in RGCs. The reversal potential of GABA-induced currents (E(GABA)) in RGCs gradually shifted from depolarized to hyperpolarized levels during 2-4 dpf and the excitation-to-inhibition (E-I) switch of GABAergic action occurred at around 2.5 dpf when RGCs became light sensitive. Meanwhile, GABAergic transmission upstream to RGCs also became inhibitory by 2.5 dpf. Furthermore, down-regulation of the K(+)/Cl() co-transporter (KCC2) by the morpholino oligonucleotide-based knockdown approach, which shifted RGC E(GABA) towards a more depolarized level and thus delayed the E-I switch by one day, postponed the appearance of RGC LERs by one day. In addition, RGCs exhibited correlated giant inward current (GICs) during 2.5-3.5 dpf. The period of GICs was shifted to 3-4.5 dpf by KCC2 knockdown. Taken together, the GABAergic E-I switch occurs coincidently with the emergence of light responses and GICs in zebrafish RGCs, and may contribute to the functional development of retinal circuits.

Figure 1. The development of light-evoked responses in zebrafish RGCs

A, diagram of in vivo whole-cell recording in intact zebrafish larvae. Top: bright-field image of whole-body morphology of a 3 dpf larva with removal of the lens (arrow). The recording pipette approached the retina under visual control. Bottom: infrared DIC image of the surface of the ganglion cell layer, in which in vivo whole-cell recording was performed on a RGC (arrowhead). B, representative traces showing light-evoked responses (LERs) of RGCs from a 2, 2.5, 3 or 5 dpf larva in response to the onset of visual stimuli (grey step). Conventional whole-cell recording was performed and the cell was held at the equilibrium potential of Cl⁻ (−60 mV). Five traces from one RGC at each developmental stage are overlaid. Traces with or without LERs are shown in black or grey, respectively. C, developmental changes in the percentage of RGCs exhibiting LERs recorded from 2–9 dpf larvae. Numbers in parentheses represent the number of cells exhibiting LERs/the total number of RGCs recorded. D, developmental changes in the onset latency of RGC LERs calculated from 63 cells. Each open circle represents the data obtained from one RGC and each filled square represents the mean latency at each developmental stage. For clarity, the mean latency is displaced slightly. The values are represented as mean ± S.E.M.

Figure 3. Developmental shift of E_GABA in zebrafish RGCs

A, GABA-induced currents recorded from two RGCs by using gramicidin perforated-patch whole-cell recording (left: 2.5 dpf; right: 6 dpf) when the cell was held at different membrane potentials (in mV) shown at the left of the traces. The currents were evoked by focal application of GABA (black squares) near RGC soma. The mean peak current at each holding potential was measured in the time window covered by the grey bar. B, plots of mean peak current against holding potential. The data were obtained from the cells shown in A. E_GABA was then calculated by fitting the curve with linear regression. C, developmental changes in mean E_GABA (black squares) of RGCs during 2–6 dpf. The filled triangles and dashed line show the mean resting membrane potential (V_RMP) of RGCs. The numbers in the parentheses represent the number of RGCs examined for E_GABA. For comparison, the developmental change in the percentage of RGCs exhibiting LERs (Fig. 1C) is shown by the grey symbols and line. D, developmental increase in the percentage of RGCs exhibiting hyperpolarizing response to GABA puff. Numbers in the parentheses represent the number of cells with hyperpolarizing response/the total number of RGCs recorded. The inset shows a GABA puff-induced hyperpolarizing response under zero holding current clamp in a 3 dpf RGC by using gramicidin perforated-patch recording.

Figure 2. Classification of zebrafish RGCs during development

A–C, representative LERs of an ON–OFF (A, 5 dpf), ON (B, 6 dpf) or OFF RGC (C, 6 dpf) in response to a 2 s flash (bottom) when the cell was held at the equilibrium potential of Cl⁻ (−60 mV) (top) or recorded at current-clamp mode (middle) by using conventional whole-cell recording. D, developmental changes in the proportion of three physiological subtypes of RGCs recorded from 2.5 to 9 dpf. The value in the histogram represents the number of RGCs examined. E–G, two-photon fluorescent images showing dendritic arborization of an ON–OFF (E), ON (F) or OFF RGC (G), of which LERs was first recorded. The arrowhead indicates RGC axon. The dashed lines represent the border of two sublaminae in the inner plexiform layer (IPL). The borders were discriminated in bright-field images. H, bright-field image of the same retina as E. The box indicates the area in E. a: sublamina a, b: sublamina b, GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.

Figure 5. Enhancement of light-evoked excitatory synaptic response in RGCs by blockade of GABAergic function

A, two examples showing that bath application of picrotoxin (100 μm) enhanced both ON and OFF light-evoked excitatory synaptic currents. Conventional whole-cell recording was performed and RGCs were held at −60 mV. B, summary of all experiments. Only ON responses were analysed. Each open circle represents the data obtained from one RGC and each filled square represents the mean value at each developmental stage. For clarity, the mean value is displaced slightly.

Figure 4. E_GABA of RGCs with and without light response at 2.5 and 3 dpf

Based on the appearance of LERs, RGCs recorded at 2.5 and 3 dpf were classified into two groups. Dark grey bars: data from RGCs without LERs; light grey bars: data from RGCs with LERs. The values in the bars represent the number of RGCs examined. *P < 0.01, **P < 0.001.

Figure 6. KCC2 down-regulation delays GABAergic E–I switch in RGCs

A, GABA-induced currents recorded from a RGC in a control (left) or KCC2 MO-injected (right) 3 dpf zebrafish larva. Gramicidin perforated-patch whole-cell recording was performed. B, plots of mean peak current against holding potential. The data were obtained from the cells shown in A. E_GABA was calculated by fitting the curve with linear regression. C, effects of KCC2 down-regulation on the developmental changes in RGC E_GABA (continuous line) and V_RMP (dashed line) during 3–5 dpf. For comparison, the developmental changes in E_GABA and V_RMP of wild-type RGCs (Fig. 3C) are shown here. The total number of RGCs examined was 82 in control MO group and 144 in KCC2 MO group. *P < 0.01, **P < 0.001.

Figure 7. KCC2 down-regulation delays the acquisition of RGC light response

A, representative LERs of five RGCs in control MO- (top) and KCC2 MO-injected (bottom) 3 dpf larvae. Conventional whole-cell recording was performed and RGCs were held at the equilibrium potential of Cl⁻ (−60 mV). B, effects of KCC2 down-regulation on the developmental changes in the percentage of RGCs exhibiting LERs. For comparison, the developmental changes of LERs in wild-type RGCs (Fig. 1C) are also shown. Numbers in the parentheses represent the number of cells exhibiting LERs/the total number of RGCs examined in KCC2 and control MO groups.

Figure 8. KCC2 down-regulation delays the onset and offset of giant inward currents in RGCs

A, dual whole-cell recording (at −60 mV) from two nearby RGCs from a 3 dpf (78 hpf) larva, showing correlated periodic giant inward currents (GICs). Middle traces: one GIC in the boxed region of left traces at a higher time resolution. Right traces: the rising phase of the GIC at a higher time resolution. B, representative spontaneous GICs of RGCs in control MO-injected zebrafish larvae, showing the presence of GICs at 3 dpf (top: 72 hpf; middle: 78 hpf) but not at 4 dpf (bottom: 100 hpf). C, representative spontaneous GICs of RGCs in KCC2 MO-injected zebrafish larvae, showing the presence of GICs at 3 and 4 dpf (middle: 78 hpf; bottom: 102 hpf) but not at early 3 dpf (top: 72 hpf). D, summary of all experiments on the appearance of GICs, which were recorded from RGCs in wild-type, control MO- and KCC2 MO-injected animals aged from 2.5 to 4.5 dpf (60 to 108 hpf). Each circle or cross indicates one RGC exhibiting GICs or no GICs, respectively.

Figure 9. Schematic of developmental events during the formation of retinal functional circuits

The schematic shows the temporal sequence of the E–I switch of GABAergic action, the appearance of RGC LERs, and the onset and offset of RGC GICs in control (top) and KCC2 MO-injected (bottom) zebrafish larvae during 2–5 dpf. Accompanying KCC2 down-regulation, all the developmental events mentioned above are delayed.